1. Technical Field
This application relates to a method of activating PPARxcex3 nuclear receptor controlled target genes in vivo and screening for PPARxcex3 ligands.
2. Background of the Invention
The thiazolidinedione class of antidiabetic drugs represent one of the few treatments of diabetes that alleviate insulin resistance, hyperglycemia and hyperlipidemia in patients with NIDDM. Thiazolidinediones are ligands for peroxisome proliferator activated receptor-xcex3 (PPARxcex3), a member of the nuclear receptor superfamily. This molecular linkage implies that thiazolidinediones achieve their insulin resistance effects by regulating PPARxcex3 target genes. However, the precise pathway connecting PPARxcex3 activation to insulin sensitization remains a mystery.
The peroxisome proliferator activated receptor xcex3 (PPARxcex3, Unigene # Hs. 100724) is a nuclear receptor that regulates gene transcription in response to lipid-like ligands including 15-deoxy-xcex9412,14- PGJ2, thiazolidinediones and other related analogs. PPARxcex3 ligands have principally been used clinically for the treatment of hyperlipidemia and non-insulin resistant diabetes (NIDMM) but have been implicated for potential treatment of other diseases including obesity, colon cancer, psoriasis, inflammatory bowel disease, polycystic ovary disease, cancer (including liposarcoma, breast cancer, prostate cancer) and cardiovascular disease (including atherosclerosis, foam cell formation and endothelial cell dysfunction). Thus, the identification of PPARxcex3 ligands has important clinical implications. In particular, the target tissue for PPARxcex3 action is unknown. Therefore, it is unclear which PPARxcex3 target genes contribute to the normalization of insulin response.
Transgenic mice have been developed that express a constitutively active form of PPARxcex3 in either brown fat, white fat, skeletal muscle or liver. Each mouse line will be evaluated for parameters of insulin responsiveness under normal and diabetogenic conditions. This will allow us to determine whether activation of PPARxcex3 target genes in any one (or combination) of tissue(s) is sufficient to relieve insulin resistance in vivo.
Molecular cloning studies have demonstrated that nuclear receptors for steroid, retinoid, vitamin D and thyroid hormones comprise a superfamily of regulatory proteins that are structurally and functionally related. Nuclear receptors contain a central DNA binding domain that binds to cis-acting elements (response elements) in the promoters of their target genes. Once bound to a response element, nuclear receptors activate transcription of specific genes through their conserved C-terminal ligand binding domains which bind hormones with high affinity and specificity. The ligand binding domain is a complex entity containing several embedded subdomains. These include a C-terminal transactivation function and a dimerization interface. Binding of the specific ligands to the nuclear receptor results in a conformation change that promotes interactions between the transactivation domain and several transcriptional co-activator complexes. These complexes destabilize chromatin and activate transcription. Through this mechanism, nuclear receptors directly regulate transcription in response to their specific ligands.
An important advance in the characterization of this superfamily of regulatory proteins has been the discovery of a growing number of gene products which possess the structural features of hormone receptors but which lack known ligands. These are known as orphan receptors, which like the classical members of the nuclear receptor superfamily, possess DNA and ligand binding domains. They are believed to be receptors for yet to be identified signaling molecules.
The peroxisome proliferator activated receptors (PPARs) represent a subfamily of structurally related nuclear receptors. Three subtypes have been described: PPARxcex1, xcex3, and xcex4. The DNA recognition sequences for all PPAR subtypes are composed of a directly repeating core-site separated by 1 nucleotide. A common recognition sequence is XXXAGGTCAXAGGTCA (SEQ ID NO:1), however, the core-site (AGGTCA; SEQ ID NO:2) is variable and may change by one or more nucleotide. To bind DNA, PPARs must heterodimerize with the 9-cis retinoic acid receptor (RXR).
The xcex1 subtype of PPAR is expressed at high levels in liver and was originally identified as a molecule that mediates the transcriptional effects of drugs that induce peroxisome proliferation in rodents. In addition, PPARxcex1 binds to and regulates transcription of a variety of genes involved in fatty acid degradation (xcex2- and xcfx89-oxidation). Mice lacking functional PPARxcex1 exhibit decreased xcex2-oxidation capacity and are incapable of increasing this capacity in response to PPARxcex1 ligands). Further, these mice inappropriately accumulate lipid in response to pharmacologic stimuli and develop late-onset obesity. Taken together, these data indicate that PPARxcex1 acts as both a sensor and an effector in a feedback loop that induces lipid catabolism in response to fatty acid signals.
In contrast to PPARxcex1, the xcex3 subtype of PPAR plays a critical role in the opposing process of fatty acid storage. PPARxcex3 is expressed at high levels in adipocytes where it has been shown to be critical for adipogenesis. Indeed, forced expression of PPARxcex3 in fibroblasts initiates a transcriptional cascade that leads to the expression of adipocyte-specific genes and ultimately to triglyceride accumulation. This implies that signals which modulate PPARxcex3 activity may serve a primary role in regulatory energy homestasis.
PPARxcex4 is ubiquitously expressed and binds several polyunsatured fatty acids as well as carbaprostacyclin, a synthetic analog of PGI2. PPARxcex4 has been suggested to contribute to the control of embryo implantation and the inhibitory effects of non-steroidal anti-inflammatory drugs on colon cancer.
That PPARs regulate lipid homeostasis implies that putative PPAR ligands represented endogenous regulators of lipid homeostasis. One ligand for PPARxcex3 has been identified 15-deoxy-xcex9412,14-prostaglandin J2 (15d-J2). The thiazolidinedione class of anti-diabetic agents mimic 15d-J2, acting as potent ligands. Moreover, the potency of thiazolidinediones as antidiabetic agents correlates with their in vitro affinity for PPARxcex3. Forman et al., Cell 83:803-812 (1995); Wilson et al., J. Med. Chem. 39:665-668 (1996). These data suggest that PPARxcex3 mediates the antidiabetic activity of these compounds.
Several other studies have shown that thiazolidinediones simultaneously promote insulin sensitization and increases in adipose cell number/mass in rodent models of NIDDM. Similarly, a genetic analysis suggested a link between obesity and a lower degree of insulin resistance in humans harboring an activating mutation in the N-terminus PPARxcex3. Ristow et al., N. Engl. J. Med. 339:953-959 (1998). That activation of PPARxcex3 can induce adiopogenesis in cell culture as well as promote insulin sensitization in vivo appears paradoxical given the epidemiological studies that link weight gain and obesity to NIDDM. However, like the pharmacologic data in rodents, this genetic data suggests that PPARxcex3 activation dissociates adipogenesis from insulin resistance.
Thiazolidinediones reverse insulin resistance in skeletal muscle, adipose tissue and hepatocytes. Komers and Vrana, Physiol. Res. 47(4):215-225 (1998). An increase insulin responsiveness is accompanied by a normalization of a wide range of metabolic abnormalities associated with NIDDM, including elevated levels of glucose, insulin, triglycerides, free fatty acids and LDL-cholesterol. Thiazolidinediones do not promote insulin secretion nor do they act as hypoglycemic agents in non-diabetic animals, implying that PPARxcex3 regulates genes that reverse a critical step in the development of insulin resistance.
Several interesting hypotheses have been proposed to explain what causes insulin resistance and how PPARxcex3 activation reverses this process. Insulin resistance may result from increase in circulating levels of free fatty acids. McGarry, Science 258:766-770 (1992). If this is the case, PPARxcex3 activation would be predicted to reverse insulin resistance by promoting an increase in fatty acid storage in adipocyes. However, this does not account for the observation that free fatty acids are not elevated in all diabetic models and that a lowering of fatty acids using other treatments is not sufficient to promote insulin sensitization. Alternatively, Spiegelman and colleagues have suggested that insulin resistance results from an increased production of TNFxcex1 in the adipose tissue of diabetics. Uysal et al., Nature 389:610-614 (1997). Under this theory, PPARxcex3 ligands act by blocking the TNFxcex1-mediated inhibition of insulin signaling, however this is not consistent with all models of NIDDM. How PPARxcex3 normalizes insulin resistance thus remains unclear.
PPARxcex3 is expressed at high levels in both brown (BAT) and white adipose tissue (WAT). In vivo administration of PPARxcex3 ligands have been shown to increase the size of both fat depots. In principle, therefore, both of these tissues could be involved in regulating insulin responsiveness. Transgenic mice with decreased levels of both BAT and WAT may be made by expressing the diphtheria toxin in these tissues using the adipocyte specific aP2 promoter. Burant et al., J. Clin. Invest. 100:2900-2908 (1997). By 8-10 months of age these mice apparently lack subcutaneous or intra-abdominal triglyceride-containing adipose tissue. The loss of triglyceride containing cells was accompanied by a progressive increase in insulin resistance and the development of diabetes. Despite the apparent loss of adipose tissue, administration of thiazolidinediones to these mice still resulted in insulin sensitization. These findings indicate that the antidiabetic action of thiazolidinedione occurs independently of thiazolidinedione-induced increases in adipocyte triglyceride content, and perhaps independent of adipose tissue. Burant et al., J. Clin. Invest. 100:2900-2908 (1997). However, this may depend on how adipocyte is defined. It is known that PPARxcex3 is induced early in the course of adipogenesis and that PPARxcex3 expression is required for subsequent activation of the aP2 promoter in adipocytes. This transcriptional cascade is followed by massive triglyceride accumulation. The strategy employed by Graves and colleagues for generation of xe2x80x9cfat-freexe2x80x9d mice depends on expression of a toxic transgene under control of the fat-specific aP2 promoter. However, since the expression of the toxic transgene in fat requires the presence of PPARxcex3, these mice should possess adipocyte-precursors which express PPARxcex3 in the atrophic remnants of adipose tissue. Thus, it may be more precise to state that thiazolidinedione action is independent of mature adipose tissue. Previous studies have not been designed to address the issue of whether the antidiabetic effects of thiazolidinediones are mediated by cells of the adipocyte lineage.
PPARxcex3 may also be expressed in skeletal muscle and in the liver but its level of expression is at least 10-fold lower in these tissues than in fat. The analysis of PPARxcex3 expression in skeletal muscle has been complicated by the presence of fat cells that interdigitate among the myocytes. Since PPARxcex3 is expressed at high levels in fat, it is possible that PPARxcex3 transcripts seen on northern blots are derived from the contaminating fat cells. Immunohistochemical assays with PPARxcex3-specific antibodies have shown that PPARxcex3 is expressed in myocyte nuclei at low levels. Despite the ability of thiazolidinediones to promote glucose uptake sensitization of skeletal muscle in vivo, these compounds had no detectable effect on glucose uptake after a five-hour exposure. Since the antidiabetic effects of thiazolidinediones are observed only after 1-2 weeks of treatment, a longer duration of exposure may be required to elicit the antidiabetic response, however it is difficult to maintain phenotypic myocytes in culture for this length of time. For similar reasons, it is not clear whether the liver is a direct target for the antidiabetic effects of thiazolidinediones.
xe2x80x9cKnockoutxe2x80x9d mice lacking the PPARxcex3 gene have an embryonic lethal phenotype. Thus, these mice are not useful in studying the effects of PPARxcex3 in the adult animal. In principle, it might be possible to bypass the embryonic lethal phenotype by generating tissue-specific knockouts of PPARxcex3. In practice, this approach has been complicated by the need to express sufficient quantities of the cre-recombinase in the target tissue. Assuming these technical difficulties can be overcome, the resulting mice would be useful in an analysis of the physiological consequences resulting from the loss of PPARxcex3 function. In any case, these mice would not be useful to study the consequences of PPARxcex3 activation. A method of studying what the effects would be in individual tissues upon activation of PPARxcex3 by a drug, or the like would be enormously useful.
Numerous screening approaches have been established to identify ligands (agonists and antagonists) for PPARxcex3. All rely on the observation that the affinity of ligand binding to nuclear receptors is determined by the receptor itself, or by a nuclear receptor dimer. Such screening methods do not take into consideration any other factors which may affect the affinity of PPARxcex3 for its ligands, either qualitatively or quantitatively, in vivo. Therefore, a screening method which is aimed at discovering novel PPARxcex3 ligands under different conditions which mimics more closely some in vivo conditions would be very useful.
Accordingly, this invention provides a method of identifying nuclear receptor controlled genes in a specific tissue of an animal, which comprises providing an expression vector containing a constitutively active nuclear receptor gene which is fused at the N-terminus to the transcriptional activation domain of the Herpes viral VP126 protein, and is linked to a promoter element which drives tissue-specific expression, transferring the constitutively active nuclear receptor gene to the animal, expressing the constitutively active nuclear receptor gene in the animal, determining the level of expression of candidate target genes of the nuclear receptor in the tissue, and identifying genes which exhibit altered expression.
In another embodiment, the invention provides a method of modulating the in vivo expression of nuclear receptor controlled genes in a specific tissue of an animal, which comprises providing an expression vector containing a constitutively active nuclear receptor gene which is fused at the N-terminus to the transcriptional activation domain of the Herpes viral VP126 protein, and is linked to a promoter element which drives tissue-specific expression, transferring the constitutively active nuclear receptor gene to the animal, and expressing the constitutively active nuclear receptor gene in vivo in the animal.
In yet another embodiment, the invention provides a transgenic non-human animal harboring a constitutively active nuclear receptor gene which is fused at the N-terminus to the transcriptional activation domain of the Herpes viral VP126 protein, and is linked to a promoter element which drives tissue-specific expression.
In yet a further embodiment, the invention provides a method of screening for compounds which bind to PPARxcex3 which comprises including a coactivation protein PBP, and an improvement to a method for screening for compounds which bind to PPARxcex3 which comprises including PBP during binding of ligands to PPARxcex3.